Sutherlandia frutescens (L.) R. Br is endemic to Southern Africa where it has been traditionally used for cancer and diabetes. In recent times it has been marketed for its reputed (but not proven) anticancer, antidiabetic and anti-HIV properties. Little is known about the mutagenic and antimutagenic potential of extracts and common marker compounds of Sutherlandia frutescens. Therefore this study aimed to investigate the putative efficacy and possible long-term adverse effects of using this herb.Ethylacetate (EA) and 50% Methanol (MeOH) extracts were screened for mutagenic and antimutagenic activity using the Ames assay utilising TA97a, TA98, TA100 and TA102 in the presence and absence of metabolic activation. Four compounds, L-arginine, L-canavanine, GABA and D-pinitol known to occur in sutherlandia were also included. The total polyphenolic content of the both extracts was determined using the Folin-Ciocalteau method and FRAP and ABTS were used to determine the anti-oxidant potential of the extracts.The extracts and the standards did not show any cytotoxicity except in TA97a. The EA extract exhibited antimutagenicity against all the bacterial strains at all concentrations tested. The MeOH extract showed both pro-mutagenic and antimutagenic activities with 2-acetamidofluorene and aflatoxin B1 in the presence of metabolic activation of TA98 and TA100, respectively. All compounds, except L-canavanine exhibited antimutagenic activity against all strains. L-canavanine, on the other hand showed co-mutagenicity with 9-aminoacridine on TA97a, at all test concentrations. The extracts and pure compounds exhibited their antimutagenic activity in a dose response manner. L-arginine and GABA showed an some antimutagenic response. EA extract had three times the total phenolic content (12.56 μg GE / mg) observed in the MeOH extract. There was correlation between total phenolic content, antioxidant potential and antimutagenicity.Both extracts exhibited a protective effect, with the EA extract exhibiting greater potency. L-canavanine acted as a co-mutagen in a dose response manner without metabolic activation. It is suggested that the EA extract be priotized for future development work as it showed a better risk profile and activity. Southern Africa boasts a rich floral diversity which is utilized in traditional herbal medicine. It is estimated that 27–30 million South Africans rely on traditional medicine for their primary health care needs , 2. TherSutherlandia frutescens (eq. Lessertia frutescens) (L.) R. Br.  also known locally as cancer bush, kankerbos (Afrikaans) and unwele (Zulu), is widely marketed in South Africa and elsewhere as sutherlandia tablets or as a leaf decoction or infusion. It is reputed, by various ethnic groups including the Zulus, Khoi-San and Xhosas, to treat a number of different diseases including diabetes mellitus, cancer, stomach complaints, topical wounds, gonorrhea and syphilis [S. frutescens has been attributed to the presence of various compounds including L-canavanine, D-pinitol, gamma (γ) aminobutyric acid (GABA) [syphilis , 7, stresyphilis , 9. In msyphilis –13. Chinsyphilis  showed tsyphilis  showed td (GABA)  and cycld (GABA) . In spitd (GABA) . FurtherThe present study investigated the mutagenic and antimutagenic potential of sutherlandia and its known constituents i.e. L-arginine, asparagine, GABA, L-canavanine, and pinitol Fig. . L-arginL-canavanine is a non-proteinogenic amino acid present in most legumes. It is a potentially toxic isomer and antimetabolite of L-arginine . Ames etGABA is the major inhibitory neurotransmitter in the CNS. Its low levels in the cerebrospinal fluid have been linked to epilepsy, schizophrenia and Alzheimer’s disease  and bipoS. frutescens. In view of the widespread use and therapeutic and/or toxic potential of sutherlandia, the present study investigated its cytotoxic, mutagenic and antimutagenic potential. This was done to understand how sutherlandia extracts and pure compounds exert their putative chemopreventive properties and if their use may be deleterious at cellular level. In addition, the polyphenolic content was assayed as this is so often linked to antioxidant activity which in turn is linked to antimutagenic activity. These polyphenolic compounds are compounds found in nature and abundant in plants and have one or more hydroxyl groups attached directly to an aromatic ring or a benzyl ring [There has been considerable interest in testing for mutagenicity and antimutagenicity  of tradizyl ring .Sutherlandia frutescens was purchased from a commercial phytomedicine supplier Parceval Pharmaceuticals (Pty) Ltd, Wellington, R.S.A. The batch number was 1,009,054 and voucher specimen (dated 20.11.2006) is deposited in the company’s herbarium in Wellington, Cape Town. The four pure compounds , 9-aminoacridine (9-AA), as well as nicotine adenine dinucleotide phosphate (NADP) and glucose-6-phosphate (G-6-P) were obtained from Sigma-Aldrich, South Africa and cumoyl hydroperoxide (CHP) was purchased from Merck Chemicals (Pty) Ltd, Darmstadt, Germany. Bacto agar and Nutrient Broth No. 2 were purchased from Difco Laboratories , Oxoid , D-Biotin and L-(−)-Histidine were purchased from ICN Biomedicals Inc.  and Merck Chemicals (Pty) Ltd,  respectively. The aroclor-1254 was obtained from Monsanto, St Louis, USA. The Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 were obtained from Prof B. N. Ames, Berkeley University, CA, USA.The mutagenic compounds i.e. 2-acetamidofluorene (2-AAF), aflatoxin BSutherlandia (100 g) was extracted with 3 × 600 ml of ethylacetate (EA) or 50% methanol (MET) in a Polytron PT 3100 blender  between 8000 and 8500 rpm for about 25 min. The pooled extracts were then centrifuged at 3000 rpm for 10 min in a Sorvall® Refrigerated Centrifuge , filtered through a Whatman No. 1 filter paper  and then dried on a rotary evaporator , at about 50 °C. All crude extracts were kept desiccated at 4 °C. In the case of aqueous methanol, freeze-drying was done after removing the methanol.w/w) whilst the 50% MeOH solution extract was diluted to 10, 25 and 50% (w/w). The pure compounds were each dissolved and diluted to 1, 2.5 and 5% (w/w) concentrations resulting in the following molar concentrations: L-arginine ; L-canavanine ; D-pinitol ; and GABA . In all cases the concentrations were determined by toxicity exhibited in the dose finding assays.Stock solutions of the crude extracts and pure compounds were prepared and subsequent dilutions made in 100% DMSO before conducting all the assays. In all cases, preliminary sub-toxic dose finding was done. The EA extract was diluted to 5, 10 and 20% (1 (AFB1) and cumoyl hydroperoxide (CHP) respectively. Tests were done in the presence and absence of metabolic activation S9 from rat liver [The strains selected for the study were TA97a (to induce deletion mutations), TA98 (for frame-shift mutations), TA100 (for base-pair substitution mutations) and TA102 . Diagnostic mutagens were 9-aminoacridine (9-AA), 2-acetamidofluorene (2-AAF), aflatoxin Bat liver , 37, 38.Salmonella mutagenicity assay [The ethyl acetate and aqueous methanol extracts were first tested for cytotoxicity using the standard plate incorporation method in the presence and absence of metabolic activation with S9 . The assty assay . MutagenThe antimutagenicity assay was conducted with the respective diagnostic mutagens for each strain in the presence of different dilutions of the extracts or pure compounds. Five replicates were used for each concentration and experiments were repeated at least once. The percentage inhibition was calculated as described previously .2 CO3 solution (75 g/ L) was added, after 120 min standing in dark, the optical density was measured at 760 nm against a blank. The TP was calculated on the basis of the calibration curve of gallic acid and expressed as gallic acid equivalents (GAE), in milligrams per gram of the sample.The total polyphenolic (TP) content of all extracts was done following the Folin-Ciocalteau (F-C) method by  with mod3 in distilled water and 100 mL of the acetate buffer prior to use. Extracts were diluted in acetate buffer . A standard curve was generated using a 5 mM stock of (±)-6-Hydroxy-2, 5, 7, 8-tetramethylchromane-2-carboxylic acid (Trolox) dissolved in ethanol. The FRAP reagent (180 μL) was added to 20 μL of the sample/standard and incubated at 37 °C for 4 min and the absorbance was measured at 592 nm. Concentration for each sample was measured in duplicate, expressed as μmol trolox equivalents (TE) per gram of extract.The ferric reducing antioxidant power (FRAP) of the extracts and samples was determined according to the method of . The FRAThe ABTS radical scavenging activity of the extracts and samples was determined according to the method of . The 2,2All individual groups were independent and tested for normality using the Kolmogorov-Smirnof Test. Levene’s Test was used to determine whether the groups had equal variances. Significant group differences were determined by the F-test  or the Welch Test , while the post-hoc Tukey Test to separate means.P < 0.05 indicated significant group differences.The Student’s Paired-sample Test was used to test for group differences when there were only two groups, with the Pooled method for groups with equal variances or the Satterthwaite method for groups with unequal variances. The effects of sutherlandia extracts and compounds in antimutagenic and mutagenic assays are summarized in Tables 1, CHP and 9-AA 1 – this was exhibited by the negative percentage found, implying that the number of revertants was higher than that of the positive control (Table Pro-mutagenic activity of the MeOH extract (50%) in the presence of S9, against 2-AAF and AFB(ii)Salmonella strains .Gomes-Carneiro et al.  demonstrSutherlandia frutescens. Both extracts exhibited a protective effect, with the EA extract exhibiting a greater potency than the MeOH extract. Pro-mutagenic activity of the latter was observed, in the presence of S9 against 2-AAF and AFB1 at the highest concentration level (50%). At the 25 and 10% concentrations however, the MeOH extract exhibited an antimutagenic response against both mutagens. An apparent stabilization of the S9 in the mutagenicity test could have contributed to the enhanced metabolic activation recorded at the higher concentration of the extract. This biphasic mode of action has been reported in the past for many natural compounds /extracts including flavonoids [In this study mutagens with different chemical structures and mechanisms of action were used to determine the protective effect of extracts of avonoids  which haon level %. At theavonoids . An inteavonoids , 54.1 in the presence of S9 is of interest. It is not clear whether any of the known compounds tested, specifically L-canavanine, did play a role in the pro-mutagenic effects of the MeOH extract. This is based on the fact that L-canavanine is co-mutagenic against TA97a in the absence of S9 but the crude MeOH extract (50%) is pro-mutagenic against both TA98 and TA100 (in the presence of S9). This could be due to the fact that L-canavanine is less co-mutagenic in combination with the other amino acids and/or that other compounds in the MeOH extract are responsible.EA extracts showed superior antimutagenic activity in all the tester strains and can also be deemed to be more potent as activity was at lower concentrations compared to MeOH. The higher antimutagenic effect and higher antioxidant activity of the EA extract can be attributed to the higher total phenolic content. Total polyphenol content correlated with superior antioxidant activity as would be expected while it also seems to be associated with the increased anti-mutagenic effects in this case. The co-mutagenicity exhibited by L-canavanine with 9-AA in the absence of S9 and the pro-mutagenic response of the highest concentration of the MeOH extract with 2-AAF and AFBEdenharder et al.  have demw/w) methanol crude extracts of sutherlandia. They obtained much higher inhibitions against TA98 and TA100 in contrast to lower inhibitions with the non-polar EA extract in this study. This study was more comprehensive in that it tested two extracts and four compounds against all four commonly used strains. EA showed a higher activity against TA98 and TA100 respectively in the presence of S9 than in the previous study by [Elgorashi et al. , 35 havestudy by . Howeverstudy by .The activity of the four compounds tested has not been reported previously. A typical dose response indicating an apparent saturation effect was observed with all the compounds. L-canavanine showed co-mutagenic rather than pro-mutagenic effects. A pro-mutagen is a synergistic agent that activates mutagenicity at low concentrations and later becomes antimutagenic at higher concentrations , 52. A cNo amino acids were found/and or observed in the EA extract, as confirmed by the TLC which we developed with ninhydrin spray reagent (results not shown). These compounds being amino acids were observed (through TLC) to be present in the 50% aq MeOH extract. L-canavanine, L-arginine, GABA and D-pinitol are extractable with more polar solvents (water and/or methanol or a mixture of both). This was confirmed by TLC. The absence of L-canavanine may explain in part the absence of pro-mutagenicity and/or co-mutagenicity in the EA fraction. The antimutagenic activity exhibited by the EA extract is therefore not due to the amino acids tested (as these were absent in this extract), but may be due to the presence of other compounds such as the less polar sutherlandosides and phenolic glycosides. Hatami et al.  showed tSalmonella strains, in the Ames assay is a positive step towards determining their safety [Plants play a major role in chemoprevention, which is recognized as a plausible and cost-effective approach to reduce cancer morbidity and mortality by inhibiting precancerous events before induction/activation of the clinical stages , 62. In r safety . Howeverr safety . Those wThe Ames assay was used in this study as it is reliable, quick and easy. This test is used to screen for possible carcinogens and mutagens. However, if a substance is screened and does not give a mutagenic response, it only suggests that the substance is not mutagenic to the particular bacterial strain it is tested against and for the genetic endpoint tested . GeneralS. frutescens crude extracts and comparing its polar and non-polar extracts as well as pure compounds. This study has at least in part, explained the pharmacological potential of non-protein amino acids from sutherlandia and that of organic and aqueous extracts. Only L-canavanine showed a co-mutagenic effect with the indirect mutagen 9-AA but it is not clear whether it is responsible for the pro-mutagenic activity of the MeOH extract with 2-AAF and AFB1 in the presence of S9. The practical implications are that formulations of aqueous sutherlandia preparations should contain little or no L-canavanine because of the potential for cellular genotoxicity shown here while formulations using less polar solvents may have a better risk profile.The current study is the first report on the mutagenicity and antimutagenicity of


Zataria multiflora (Zm-Ag.NPs) leaf extract and assessment of its apoptosis promoting effects. The Zm-Ag.NPs was characterized by UV-visible and FTIR spectroscopy, TEM, EDS, DLS, and measurement of zeta-potential. Apoptosis induction effects of Zm-Ag.NPs were assessed using acridine orange – propidium iodide (AO/PI), DAPI staining, caspase3/9 activation assay, and annexinV/PI assay. Changes in P53, matrix metalloproteinases 2 (MMPs), and vascular endothelial growth factor A (VEGF-A) genes expression were also assessed with semi-quantitative RT-PCR. The UV-visible spectroscopy results showed that the surface plasmon resonance band (SRP) for Zm-Ag.NPs was about 440 nm, also, FTIR spectroscopy indicated that plant material embedded around Zm-Ag.NPs. The TEM images of the samples revealed that the Ag.NPs varied in morphology and also, the presence of silver element was monitored with EDS. The mean size of Zm-Ag.NPs was 30 nm. The Zm-Ag.NPs reduced cell viability in a dose and time dependent manner (IC50 = 15 μg/mL). AO/PI and DAPI staining indicated chromatin fragmentation and annexinV externalization assay using flow cytometer, confirmed promotion of programmed cell death in the treated cells. Apoptosis was induced through caspase 3/9 activation pathway. This promotion of apoptosis effects is not related with P53 gene up regulation. Finally, it was found that Zm-Ag.NPs inhibited cancer cell metastasis through a decrease in MMP and VEGFA expression. Zm-Ag.NPs acts as carrier of the plant material compound, and can be applied as anticancer agents.The biosynthesis of nanoparticles is widely considered today. This investigation was aimed at the biosynthesis and coating of Ag.NPs with  In the developing world, cancer is still problematic and it is difficult to describe the right way to treat it. According to the WHO reports, cancer causes about 7.9 million deaths worldwide each year . In addi Nanotechnology is a very interesting field, which is related to the credible reports of the fabrication and use of materials with structural character between those of bulk materials and atoms with at least one dimension of nano-size. The fabrication of metal nanoparticles is an expanding field due to their ability to be used in various fields . Among them is Ag.NPs, which represent unusual biological and physicochemical activities, and, therefore, have been used in the health care industry . The spePlants and microbes are applied in the fabrication of nanoparticles. Use of plant extracts for the synthesis of nanoparticles has advantages, such as being cost-effective, eco-friendly, and also taking place of the process in one setup; also, nanoparticles act as carrier in the transfer of materials into cells . MedicinZataria multiflora, a member of the Lamiacea family is a well-known folk medicine  -2, 5-diphenyltetrazolium bromide], Acridine orange, propidium iodide, 4ʹ, 6-diamidino-2-phenylindole (DAPI), and silver nitrate were obtained from Sigma-Aldrich from England. Fetal bovine serum (FBS) and RPMI-1640 culture medium were purchased from Invitrogen, England. The high pure RNA isolation kit was purchased from Roche, Germany, and cDNA synthsised kit from fermentase. For PCR reaction, PARS TUS kit (Iran) and primer from Bioneer (Korea) were obtained. All the solutions were prepared with double distilled water and other chemicals were of analytical grade.Extraction PreparationZataria multiflora leaves were collected from Mashhad  and identified by the Herbarium Division of the Mashhad University; they were given the voucher specimen number 34516. The leaves were then washed, dried and powdered before being used for extraction. Following this step, 5 g of the leaf powder was added to 100 mL of sterile distilled water and boiled for five minutes. The extract was then filtered through a Whatman filter (paper No.1) and stored at 4 °C for further use.The green synthesis of silver nanoparticles Zm-Ag.NPs was synthesized by reducing 10 mL of silver nitrate solution (1-5 mM) with 100 to 1000 µL of the Z. multiflora leaf extract. Change in the color of the solution from pale brown to dark brown indicated the formation of nanoparticles. This process was carried out at a temperature of 40 °C and at pH 7.0. In order to eliminate any free biomass residue or unbound extract from the surfaces of the nanoparticles, the Zm-Ag.NPs was centrifuged and repeatedly washed with distilled water. Subsequently, the product was centrifuged at 9000 rpm for 30 min and dried at 45 °C.Characterization methods The UV-visible absorption spectra of the samples were measured in different concentrations of the plant extract and silver nitrate and at different time intervals using a spectrophotometer . For analysis of UV-visible spectra, 100 µL of the sample was put in a 96-well plate and read. The size of the nanoparticles was determined using DLS/zeta potential analysis . Zm-Ag.NPs at 25 °C using 0.894 cp for the viscosity of the medium, and a fixed angle = 90 for the avalanche photo diode (APD) detector and a wavelength of 657 nm for the 50 mW laser zeta-potential of Zm-Ag.NPs in water, were evaluated using CAD  zeta sizer. The TEM results  were obtained by depositing a drop of the best sample, based on the DLS results, on a copper mesh coated with a carbon film; the solvent was then evaporated. The size dispensation was obtained by image analysis using the Image J software package 24, counting at least 200 particles for significant and relevant statistics. The FTIR for the leaf of the plant extract and Zm-Ag.NPs was obtained in the range of 4000 to 400 cm.−1 with a Perkin Elmer spectrophotometer paragon 1000. The dried Zm-Ag.NPs sample was placed on a coated carbon film and examined using EDS analysis .The best sample base of the UV-visible results was selected for the DLS study. DLS was applied to assess the size of Cell culture assayMTT assay4 HeLa cells were seeded in a 96 well-plate. After 24 h the cells were treated with 5-60 µg/mL Zm-Ag.NPs for 24 and 48 h, at which point, the culture media was aspirated off, and 20 µL of the MTT solution (5mg/mL) was added, before being incubated for 4 h in a cell incubator . After this, the MTT solution was aspirated off from the wells. About 5×10The formazan (MTT metabolic product) was resuspended in 200 µL isopropanol, and a 96-well plate reader was used to read the absorbance at 570 nm. The cell viability was calculated using the following equation:Where A treated and A control are the absorbance of the treated and untreated cells, respectively  DAPI staining3 on gelatin-coated coverslips placed on tissue culture previously. The cells were treated with 15 µg/mL Zm-Ag.NPs. After 48 h 200 µL of 4% paraformaldehyde was added to the fixed cells and incubated for 20 min at room temperature. The wells were washed twice with PBS, stained with 100 ng/mL DAPI for 510 min and finally, washed with PBS, then observed using fluorescence microscopy with DAPI.The cells were cultured by adding approximately 5×10Acridin orange – propidium iodide (AO/PI) staining The seeded HeLa cells were treated with 10 and 20 µg/mL for 48 h. For the AO/PI staining, the cells were harvested and re-suspended before 10 µL of the treated cell sample, 10 µL of the AO staining solution and 10 µL of PI staining solution were combined. Then, 10 µL of the stained sample was added to a Cellometer Counting Chamber and analyzed using fluorescence microscopy . Caspase activation assay Zm-Ag.NPs for 24 h. After this, the 1-5 × 106 cells were pelleted and re-suspended in 50 μL of chilled Cell Lysis Buffer, before centrifuging for 1 min. The protein concentration was assayed using the Biuret method. For each assay, 100 μg proteins were diluted with 50 μL Cell Lysis Buffer. Finally, the DEVD-p-NA substrate was added and the samples were read at 400 or 405 nm using a microtiter plate reader . The fold-increase in Caspase 3 activity was determined by comparing with the control groups.Caspase-3 and 9 activities were assessed using the colorimetric protease assay Abcam Kit following the protocol of the manufacturer. Briefly, the cells were treated with Zm-Ag.NPs. Apoptosis was induced in the cells treated with Annexin: Determination of apoptotic and necrotic cellsZm-Ag.NPs for 48 h before harvesting and centrifuging at 200 xg for 5 min. Then, 5 µL Annexin- V-FITC labeling and 5 µL PI solution were added, before incubating for 5 min at 25 °C and analyzing using a flow cytometer . The amount of apoptotic specific hallmark externalization of PS on the HeLa cell surface was assessed using the Annexin-V-FITC staining kit (Abcam), Mannheim, Germany, according to the manufacturer’s instructions. The cells were tested with 10 and 20 µg/mL Gene expression analysisChanges in the expression of VEGF, MMP, and P-53 genes were analyzed using RT-PCR. Briefly, the total RNA of the treated MCF-7 cells was isolated using the High Pure RNA Isolation kit according to the manufacturer’s protocol . Then, the cDNA was synthesized using the ParsTous Kit, incubated at 65 °C for 5 min followed by the addition of the RT premix. 2 25 mM, dNTP, Taq DNA polymerase and the appropriate forward and reverse primers. Ultimately, RT-PCR was performed: 1 cycle at 95 °C/4 min, 35 cycles at 94 °C/30 s for denaturation, 57 °C/30 s for annealing, 72 °C/30 s for extension, and 1 cycle of 5 min at 72 °C. The primers were used as follows:The temperature of the synthesis was according to the protocol: incubation at 65 °C for 5 min followed by the addition of the RT premix, then incubation at 25 °C for 10 min, 50 °C for 60 min and 70 °C for 10 min. Finally, 2 µL of the cDNA produced was added to the 10xbuffer, MgClThe PCR products were observed by electrophoresis in a 2% agarose gel, read using a UV-detector and semi-quantitatively measured from the UV optical density of bands using ImageJ software.Statistical analysisp < 0.05 was calculated as the minimum level of significance.Statistical assessment of the data was performed using ANOVA one-way analysis. The Tukey test was applied for comparisons as a posttest with the help of SPSS software, and the results are shown as mean ± SD, and Green synthesis of Zm-Ag.NPs and characterization Zm-Ag.NPs exhibit a brown color in aqua medium. This color appears due to the excitation of the surface plasmon vibrations in the metal nanoparticles. The formation of Zm-Ag.NPs was only observed in the mixture containing the AgNO3 solution and in the leaf extracts at a maximum absorbance of 440 nm after 4 h. The color change was distinguished by visual observation in the extract of the Zm leaf when incubated with AgNO3 solution. The color intensity increased with the increased dose of the plant extract at the same duration, at 40 ºC. The color intensity was greatest when 10 mL of AgNO3 solution at 2.5 mM and 0.5 mL of the Zm leaf extract (10:0.5 v/v) were mixed together. 3 solution without the leaf extract, while 3, which did not show any significant change in color, and Zm-Ag.NPs.It is well known that UV-vis analysis of silver nanoparticlesZm-Ag.NPs and concentration of silver nitrate were noted. The concentration of silver nitrate is the first factor. When the concentration of silver nitrate increased from 1 to 2.5 Mm, the Zm-Ag.NPs absorbance value increased , were used for future characterizations.ubstrate . OverallEDS proﬁle of synthesized silver nanoparticlesZm leaves was characterized by EDS analysis, which provides evidence for the reduction of silver nitrate to elemental silver. The spectrum showed a strong silver signal together with about 3 kv with no contamination  was used in conjunction with the scanning electron microscope (SEM), providing chemical analysis in areas as small as 1 µm in diameter. EDS detects all elements except H, He, Li and Be. EDS can be performed exactly on any feature or particle seen in the SEM images and can “MAP” elements on a surface. Unknown materials can be identified and quantitative analysis can be performed. The synthesis of silver nanoparticles using the extract of mination .Zm-Ag.NPs synthesized using the extract of Zm leaves. The FTIR spectra for Zm-Ag.NPs are shown in The FTIR measurement was done to identify the probable biomolecules responsible for the efﬁcient stabilization and capping of Zm extract in the Zm-Ag.NPs as a capping agent. The Zm-Ag.NPs and plant extract FTIR spectra suggest the presence of various plant compounds, such as flavonoids and polyphenols, apart from other phytochemicals that were responsible for the formation of the Zm-Ag.NPs by reducing the Ag ions. The FTIR spectroscopic analysis confirms that the proteins present in the Zm leaf extract might act as a reducing agent, stabilize the Zm-Ag.NPs, and prevent agglomeration. Similar methods using the FTIR have been previously used to study Gnidia glauca flower extracts after bio reduction , pentagonal (arrow marked 2) and undefined (arrow marked 3) shapes.TEM is a powerful method to show the shape and size of the nanoparticles . The sizDynamic light scattering (DLS)studyZm-Ag.NPs ranged from approximately 16 to 70 nm with an average size of 30 nm. This confirms that the sample contains various sizes of nanoparticle , the values of zeta potential were equal to -10 to -40 mV. It could be concluded that epulsion .Cell cultureCytotoxity assayZm-Ag.NPs were found to have the highest toxicity against HeLa cancer cells (IC50 = 15 μg/mL). Toxicity increases over time and in a concentration dependent manner, as the IC50 values of Zm-Ag.NPs were calculated as 15 μg/mL for 24 h and 10 μg/mL for 48 h respectively (Zm-Ag.NPs (5 to 30 nm) had a stronger cytotoxic effect, as they produced greater amounts of hydrogen peroxide  of the dye was quantified using a multiwell-spectrophotometer at 570 nm. ectively . Smallerperoxide . The aveiciently .Apoptosis morphological identification with DAPI nuclear stainZm-Ag.NPs lead to a breakdown in chromatin, as shown in S. cumini extract on Dalton lymphoma cell lines were investigated and it was revealed that Ag.NPs can generate ROS that breaks down DNA, a hallmark in cells that appears to be undergoing apoptosis induction  /propodiumiudid (PI)Acridine orange (AO) is a metachromatic dye through which single-stranded (ss) stains emit green fluorescence in the interaction with double-stranded (ds) DNA, whereas for intercalation with DNA extraction, it is red fluorescence. Chromatin break down is an event of apoptosis that could be indicated with AO staining. Propodiumiodide enters into the cells that have lost their membrane integrity and indicates necrotic or late apoptosis cells .AnnexinV/PIZm-Ag.NPs on the apoptosis of HeLa cells was evaluated using the annexin V/PI double staining method. The results indicated that, following the treatment of HeLa cells for 48 h with 10 and 20 µh/mL Ag-NPs, the number of early apoptotic cells increased to 2.3 and 24.2, respectively, in 48 h. The cells in the late apoptosis stage increased to 4.5 and 67.2. The results revealed that Zm-Ag.NPs induced apoptosis in HeLa cells. Other investigations also proved that the apoptotic indexes indicated that Zm-Ag.NPs exhibited an apoptotic effect in a concentration-dependent manner  residues are found in the inner membrane of the cytoplasmic membrane in cells. During apoptosis, the PS becomes externalized. Annexin-V specifically binds to the PS, and, hence, the protein can be applied to detect apoptotic cells. Annexin-V conjugated to a fluorochrome dye is available. In this study, the effect of t manner   in the MMP-2 gene expression (mRNA level). The level of P-53 after treatment increased dramatically but was not statistically significant  are associated with cancer-cell invasion and metastasis. Tumor cells utilize the matrix degrading capability of these enzymes to spread to distant sites. They also act as regulatory molecules like adhesion molecules that modulate the bioavailability growth factors, such as VEGF to generate fragments with enhanced or reduced biological effects. In addition, MMPs are also thought to promote the growth of these tumor cells by activating cell metastasis. Of the various MMPs thought to be involved in cancer, attention has focused on the gelatinases, because (i) they are overexpressed in a variety of malignant tumors and (ii) their expression and activity are often associated with tumor aggressiveness and a poor prognosis . In thisnificant . Zm-Ag.NPs is presented using the biodegradable Z. multiflora leaf extract. The diameter of the Zm-Ag.NPs was in the range of 16 to 70 nm, as shown by the DLS. The flavonoid, terpenoid, and protein constituents, which were present in the Zm leaf extract, were the surface active molecules stabilizing the nanoparticles. The hydroxyl and carboxylate groups of the extract facilitate reduced elemental silver. This proposed mechanism is also substantiated by the FTIR data. In this study, the toxicity effect of the biosynthesis of Zm-Ag.NPs as a potential agent for chemotherapy on HeLa cancer cells was assessed. The results indicated that treatment of HeLa cells with Zm-Ag.NPs leads to an increase in the expression of P53 protein and a decrease in the expression of VEGF and MMP-2. These proteins are involved in the progress of cancer. Zm-Ag.NPs also increases the activity of procaspase-9 and caspase-3. The involvement of Zm-Ag.NPs in induction of apoptosis leads to an increase in caspase-3/9 activity. Its effect on apoptosis was further confirmed by measuring its activity and the induction of apoptosis was measured by flow cytometry. As DNA damage is a feature of apoptotic cell death, DNA breakdown was further confirmed using the DAPI staining. This Ag.NPs is a carrier of plant material such as phenolic compound that can act as anticancer agents.One-step green synthesis of


Whole-cell biocatalysts provide unique advantages and have been widely used for the efficient biosynthesis of value-added fine and bulk chemicals, as well as pharmaceutically active ingredients. What is more, advances in synthetic biology and metabolic engineering, together with the rapid development of molecular genetic tools, have brought about a renaissance of whole-cell biocatalysis. These rapid advancements mean that whole-cell biocatalysts can increasingly be rationally designed. Genes of heterologous enzymes or synthetic pathways are increasingly being introduced into microbial hosts, and depending on the complexity of the synthetic pathway or the target products, they can enable the production of value-added chemicals from cheap feedstock. Metabolic engineering and synthetic biology efforts aimed at optimizing the existing microbial cell factories concentrate on improving heterologous pathway flux, precursor supply, and cofactor balance, as well as other aspects of cellular metabolism, to enhance the efficiency of biocatalysts. In the present review, we take a critical look at recent developments in whole-cell biocatalysis, with an emphasis on strategies applied to designing and optimizing the organisms that are increasingly modified for efficient production of chemicals. Developing sustainable bio-based processes for production of fuels, chemicals, and materials is increasingly attractive due to increased concern for the environment. Chemical synthesis processes are generally high-yield, however they are often environmentally unfriendly and are associated with the production of unwanted byproducts, thus reducing efficiency and increasing downstream costs. In comparison with chemical catalysis, whole-cell biocatalysis offer some unique advantages Table  and provPseudomonas putida (for butane activation) and the RhlABC system from Pseudomonas aeruginosa (for rhamnolipid assembly) were integrated into the cell strain to result in rhamnolipid biosynthesis that used butane as the sole carbon and energy source. This approach represents a one-pot convergent total synthesis with more than 25 steps [Whole-cell catalysis approaches can broadly be classified into biotransformation  and fermentation bioprocesses. In fermentations, the products are synthesized from growth substrates via the host cells’ native metabolism and are accompanied in the fermentation broth by metabolic intermediates that make downstream processing complicated , 12. In 25 steps . Whole-c25 steps . In an e25 steps –19.Efficient whole-cell biocatalysts are very important for an economically feasible biocatalysis process with optimal titre, yield and productivity. The principles for designing whole-cell biocatalysts for bioconversions are quite different from those for designing the microbial cell factories that are used in fermentations –19. SingEscherichia coli is perhaps the most widely used microbial platform for cell factories. This dominance is largely because of its well-studied genetic background, a mature and powerful genetic toolset for metabolic engineering, and relatively well-developed fermentation processes with low-cost raw materials [E. coli seems to be the most promising method and offers the potential for large-scale and low-cost production. Consequently, this review focuses on whole-cell biocatalysis using microbial cell factories based on E. coli.aterials , 13, 16.aterials –24. The aterials , 26. Assaterials . Whole-cBiocatalysis using a single strain as the sole catalytic unit can offer a broad scope of substrates that is not limited to natural pathways and thus opens the door to versatile multistep biocatalysis. Additionally, whole-cell biocatalysts can be rationally designed and readily tailored to their specific applications. In this review, we summarize the different strategies and efforts that have been used in designing and optimizing various whole-cell biocatalysts to convert cheap feedstocks to value-added chemicals.Whole-cell biocatalysts allow for the facile implementation of enzymatic cascades that span multiple reactions, with an integrated supply of the myriad cofactors that are needed for such complex biotransformations . This inAs with any catalytic process, the cost and stability of the catalyst are highly relevant to its economical application in chemical manufacturing . The useTypical biotransformation processes comprise two stages: growth of the living “catalyst” and conversion of the substrate(s). After cells are cultured, they are harvested and washed with water or a buffer solution and suspended in the desired buffer for biocatalysis. When the cells are washed, unconsumed growth substrates and nutrients, as well as undesired metabolites that were produced during growth, are removed from the system, allowing significantly better product recovery rates and greatly simplifying downstream processing . FurtherThe construction of effective whole-cell biocatalysts requires that single or multiple enzymes are introduced into host cells to construct synthetic pathways for the conversion of the desired feedstocks into the targeted products. Metabolic engineering and synthetic biology efforts aim to enable rational design and construction of biosynthetic pathways that maximize the pathway flux to products by providing preoptimized chassis cells that enhance the production of target compounds. The strategies that are employed in the design and optimization of whole-cell biocatalysts are discussed below Fig. .Fig. 1StE. coli to generate heterologous biosynthesis pathways for the production of exogenous and non-natural molecules. Heterologous expression of genes from the designed pathway in E. coli can be controlled independent of the host’s regulatory network [Many new genes and pathways have been discovered with the recent advances in genomics, proteomics, and bioinformatics. The numerous potentially valuable enzymes and pathways that are present in nature are being identified at an increasing pace , 12, and network , resultiAlthough whole-cell biocatalysis is primarily an alternative for in vitro multienzyme cascades, a number of successful and commercially viable whole-cell biocatalysts that use single-step reactions to produce fine chemicals exist , 29. ForE. coli produced cinnamyl acetate as a result of its high transesterification activity. The conversion ratio of cinnamyl alcohol reached 94.1% at 1 h and was even higher (97.1%) at 2 h [l-carnosine production, two enzymes (DmpA from Ochrobactrum anthropi and BapA from Sphingosinicella xenopeptidilytica 3-2W4) were screened. High yields of l-carnosine (up to 71%) were obtained by optimizing relevant reaction conditions for the best-performing recombinant strain (E. coli DmpAsyn). The biocatalyst had a high stability and could be used in at least five sequential batches. In another case, whole-cell biocatalysts for γ-aminobutyric acid (GABA) production were developed by overexpressing glutamate decarboxylase . GADs from different species were expressed and screened, and the enzyme from Lactococcus lactis was chosen. The gene gadAB was subsequently deleted from the genome of the E. coli host to prevent the degradation of GABA. As a result, 614.15 g/L GABA was produced with a high molar yield (over 99%) [A whole-cell catalyst that expressed a novel esterase (EstK1) in ) at 2 h . For whover 99%) . In thisl-proline to trans-4-hydroxy-l-proline (Hyp), is a 2-oxoglutarate (2-OG)-dependent oxygenase [Dactylosporangium sp. RH1 was overexpressed in a putA-deficient E. coli mutant that cannot degrade l-proline. The co-substrate 2-OG, an intermediate of the citric acid cycle, was supplied by glucose via the central carbon metabolism of the cells. Hyp was accumulated to a final concentration of 41 g/L with a productivity of 0.41 g/L/h [l-glutamate oxidase (LGOX) catalyzes the transformation of l-glutamic acid to 2-OG along with the production of NH3 and H2O2. In order to remove H2O2, catalase was co-expressed with l-glutamate oxidase, which substantially enhanced the efficiency of 2-OG production. 77.4 g/L 2-OG with a conversion rate of 98.5% was obtained in 12 h [Proline-4-hydroxylase, which catalyses the biotransformation of xygenase . The pro41 g/L/h . In this in 12 h .l-phenylalanine (l-Phe) via the three-step Ehrlich pathway in yeast and via the phenylacetaldehyde synthase (PAAS) pathway in plants. The reconstitution of the Ehrlich pathway in E. coli introduced the enzymes that perform the necessary decarboxylation and reduction steps into the bacterial cells and resulted in 2-PE production. Approximately 96% of the final product was produced from l-phenylalanine  using the recombinant E. coli [Rosa hybrid, a pyridoxal 5′-phosphate (PLP)-dependent enzyme and endogenous alcohol dehydrogenases was introduced into E. coli to produce 2-PE. This biotransformation, which was based only on internal de novo PLP synthesis, produced 0.34 g/L 2-PE [In cases of complex multistep bioconversions, entire metabolic pathways can be transferred to the host from other organisms. The aromatic alcohol 2-phenylethanol (2-PE) is synthesized from  E. coli . In anotg/L 2-PE , 36.d-neuraminic acid (Neu5Ac) in bacteria has been identified. Although researchers have tried to metabolically engineer a corresponding E. coli strain, only 1.5 g/L Neu5Ac was obtained by fermentation [E. coli strain; this new pathway co-expressed GlcNAc 2-epimerase  from cyanobacteria and Neu5Ac aldolase  [E. coli. Ishikawa et al. constructed a recombinant E. coli N18-14 by overexpressing genes of GlcNAc 2-epimerase (slr1975) and neuB resulting in a yield of 53 g/L of Neu5Ac (2.41 g/L/h) after 22 h [E. coli co-expressing slr1975 and nanA for Neu5Ac production was reported and Neu5Ac accumulated at 59 g/L after 36 h (1.64 g/L/h) [E. coli consisting of AGE from Anabaena sp. PCC7120 and NanA from E. coli was used as whole-cell biocatalyst [The construction of de novo biosynthetic pathways denotes the assembly of genes from different non-related organisms to construct artificial pathways in the desired host. This approach enables retrosynthetic pathway design and opens the door to the development of unprecedented multistep biocatalysts. The seven-step metabolic pathway from glucose-6-p to N-acetyl-entation . In cont3, NanA) , 38, 39 3, NanA) , 25, 40.catalyst . A yieldcatalyst .E. coli co-expressing a styrene monooxygenase and two alcohol dehydrogenases was described by Liu et al. [rac)-1a, this approach was extended to other substrates. As a result, excellent enantio- and diastereoselectivity were achieved for most of the 12 substrates [Once a de novo engineered pathway has been designed and demonstrated, it can be integrated into existing platforms for secondary metabolite production in two different ways: (1) the de novo pathway can be extended by connecting it to other pathways, and (2) biocatalysts can be used to produce product derivatives by starting from chemically modified substrates. For example, Neu5Ac is the precursor of polysialic acid (PSA) and sialylated oligosaccharides. Thus, an enhanced Neu5Ac biosynthetic module can also improve production of PSA and sialylated oligosaccharides , 42. Whou et al. . With thbstrates . FinallyThe introduction or creation of biosynthetic pathways in microbial hosts has allowed the biocatalytic conversion of non-native chemicals. However, these pathways rarely function optimally when first introduced into the host organism, resulting in suboptimal yields of the desired product . Thus, sExpression of foreign pathways often results in suboptimal performance due to unintended factors such as introduction of toxic metabolites or poor expression of pathway components . The ideE. coli that consisted of AGE and NanA. NanA was identified to be the rate-controlling enzyme of the engineered pathway. Efforts had been made to alleviate the NanA bottleneck by manipulating the amount of the recombinant enzyme. When the expression of NanA increased, a nine-fold increase in Neu5Ac production was achieved [A whole-cell biocatalyst for Neu5Ac production was developed by assembling a heterologous biosynthetic pathway in achieved . For theachieved . Overexpachieved .Imbalances in pathway gene expression can lead to the accumulation of toxic intermediates or by-products, and the resulting metabolic burden on the host cells leads to suboptimal performance . TargeteE. coli strain to produce large quantities of isoprenoids [E. coli in the form of two synthetic operons, an “upper pathway” that converted acetyl-CoA to MVA and a “lower pathway” that produced dimethylallyl diphosphate (DMAPP) from MVA, that were a combination of bacterial and yeast enzymes [Enterococcus faecalis, while the lower pathway comprises the MVK, PMK, MVD, and IDI enzymes from Saccharomyces cerevisiae and Methanosarcina mazei [A very illustrative example of these approaches is the optimization of the mevalonic acid (MVA) pathway to enhance isoprenoid production. The strategies aimed to balance the pathways and eliminate the accumulation of toxic intermediates in addition to maximizing the flux towards the product. A mevalonate-based isopentenyl pyrophosphate biosynthesis pathway (MVA) was introduced into an prenoids , 50, 51. enzymes , 52. Thena mazei . Additiona mazei , 51, 53.na mazei , 51, 53.crtEBI genes from different species including Pantoea ananatis, Pantoea agglomerans, Pantoea vagans and Rhodobacter sphaeroides in combination with the host’s native IDI were assembled in E. coli to construct the basic lycopene synthesis pathway. The pathway in the library was varied by recombining four RBS targets and the crtEBI genes from different species and by varying the gene order. Strikingly, all of this variation was implemented in a single assembly step using the OLMA method. The library was subsequently directly analysed for lycopene production, and the enzymes of the lycopene synthesis pathway and their expression levels were optimized to result in a striking increase in yield from 0.14 to 15.17 mg/g DCW [Due to rapid advances in synthetic biology research, efficient techniques for the combinatorial assembly of large numbers of genes, operons and pathways are becoming readily available . DNA assmg/g DCW .nanA and nanT genes that encode the Neu5Ac aldolase and Neu5Ac transporter, respectively, abolished sialic acid catabolism, while knocking out the nanK (encoding the Neu5Ac transporter) and nanA genes prevented ManNAc and Neu5Ac from being diverted from the biosynthetic pathway [nanTEK genes of engineered E. coli were knocked out, Neu5Ac production was enhanced threefold, resulting in 173.8 mM Neu5Ac [Blocking the competing pathways that drain substrates and intermediates prevents their diversion from the desired biosynthesis pathway and usually increases the flux and final titre of the product, as expected. The removal of the  pathway , 42. WheM Neu5Ac . The comIn engineered strains, bioconversion efficiency is determined not only by metabolic flux but also by the efficient turnover of precursors . When a C-methyl-d-erythritol 4-phosphate (MEP)-dependent carotenoid biosynthesis, an imbalanced supply of glyceraldehyde 3-phosphate (G3P) and pyruvate precursors is one of the major metabolic bottlenecks, especially considering the limit of the G3P precursor’s availability. Systematic modification of targets within central metabolic pathways was conducted to promote the redistribution of metabolic fluxes towards MEP-dependent carotenoid biosynthesis. The flux to the EMP pathway was rewired towards the ED/PP pathways by knocking out phosphoglucose isomerase (PGI). Tweaking the flux at the branch point between ED and PP by overexpressing eda and fine-tuning gnd expression in a PGI-deficient strain (Δpgi) improved the G3P/Pyr supply and rebalanced precursor availability, which relieved the bottleneck. Further improvements in DXS expression led to efficient use of G3P and pyruvate in the MEP pathway and significantly increased productivity [For 2-uctivity . Thus, tA whole-cell biocatalyst is usually preferred for cofactor-dependent reactions since the inherent presence of cofactors that are generated by the host cell and the ease of their recycling greatly improve the economics of the process . In cellIncreased cofactor regeneration is traditionally accomplished using an in situ regeneration reaction Fig. a. For oxl-alanine-dependent transamination with an l-alanine dehydrogenase enabled the self-sufficient redox conversion of alcohols to the corresponding amines  were developed as a whole-cell biocatalyst to convert penicillin G to G-7-ADCA [The 2-OG-dependent oxygenases have emerged as the largest known family of nonheme-oxidase enzymes and are involved in the biosynthesis of a truly vast variety of metabolites, including materials of medicinal or agrochemical importance  . These rG-7-ADCA . The TCAE. coli, the researchers coupled bridging enzymes with transaminase and alcohol dehydrogenase so that the cofactor and redox equivalents were regenerated simultaneously and no external cofactor or redox source was required [For biosynthetic pathways that encompass both co-substrate- and redox-dependent reactions, e.g., 2-phenylethanol (2-PE) biosynthesis, cofactors such as 2-OG and NAD(P)H are required for the transamination and dehydrogenation reactions, respectively. The simultaneous regeneration of cofactors and redox equivalents remains a challenge. However, it is possible to construct a “bridge” between an amino acid and a structurally equivalent fusel alcohol by using glutamate dehydrogenase. Thus, to develop a self-sufficient cofactor system to enhance production of 2-PE in required . A self-Metabolic engineering of the chassis cell to further improve the performance of whole-cell biocatalysts may involve increasing the cells’ uptake of substrate(s), reducing substrate and product degradation, and blocking the effects of proteases to stabilize overexpressed intracellular enzymes , 66.P. putida GPo1 was reported to improve hydrophobic substrate uptake into E. coli [E. coli cells overexpressing the monooxygenase system AlkBGT and the uptake facilitator AlkL were used as a whole-cell biocatalyst to oxyfunctionalize renewable fatty acid methyl esters [GlcNAc is transported by GlcNAc-specific PTS into cells as GlcNAc-6-P and then enters the NAG pathway to be utilized as a carbon and nitrogen source. Elimination of GlcNAc-specific PTS reduced GlcNAc-related side reactions and increased Neu5Ac production by 1.28-fold . The out E. coli . E. colil esters . Howeverl esters .ampC) resulted in a 3.9-fold increase in the G-7-ADCA production over that of the parent strain [Reducing the degradation of substrate and product is important for increasing substrate utilization and thus increasing the overall conversion rate. For example, penicillin G and G-7-ADCA are susceptible to decomposition; knocking out the gene that encoded β-lactamase (t strain .Low amounts of the relevant enzymes are obtained, which leads to suboptimal performance, if the enzymes of the synthesis pathway are susceptible to protease attack . In suchTo develop an economically feasible whole-cell biocatalysis process, in addition to the rational design of whole-cell biocatalysts, it is of great importance to also optimize the entire production process to achieve economic viability. Whole-cell biocatalysis processes must, by definition, involve a growth process and a substrate conversion process. Cells are cultured, removed from the growth medium and then resuspended in different biotransformation media to convert substrates into desired products , 13. SevTo implement a whole-cell biotransformation, the substrate of interest must be transported across the cell membrane to reach the active enzyme or enzyme system. The same issues exist for the product. Even though the substrate can usually enter the cell by passive diffusion , mass trE. coli platform. As described above, advances in metabolic engineering and synthetic biology have markedly improved the productivities and yields of products that are synthesized using whole-cell biocatalysts.Whole-cell biocatalysts can convert cheap feedstocks into complex, value-added fine chemicals with a range of applications in pharmaceutical and chemical industries. What is more, efficient whole-cell biocatalysts can increasingly be rationally designed. The present work reviews strategies for the metabolic engineering of whole-cell biocatalysts based on the well-established Despite the advantages outlined in this article, there are certain limitations that should be considered. The mass transport barrier that the cell membrane represents is one limitation. Cells often have a specific system to transport compounds in or out of the cell. Enhancing the transport system may help the transportation of substrates and products. Another method that can be used to improve substrate transfer across cell walls and membranes is to increase their permeabilization level by chemical means. However, these methods may damage the cell integrity and cause leakage of cellular components, complicating downstream process engineering; therefore, the best conditions for permeabilizing cells should be determined. Second, metabolic engineering strategies that are used in rational strain design involve the overexpression, deletion or down-regulation of genes in their native metabolic pathways , 71, 72.Whole cell biocatalysis has been successful in bioconverting non-native substrates into target products. However, challenges still remain when whole-cell biocatalysis uses glucose as a substrate for fine chemical production. Although cells are in a resting state in the conversion stage, the enzymes of central metabolism are still active; thus, the introduced biosynthesis pathways compete with the native enzymes of the central pathways for substrates and energy. Metabolically engineering targets in the central metabolic pathways would affect cell physiology and lead to fluctuations in cell growth. Redirecting fluxes of central metabolism to cell growth or to biosynthesis pathways through dynamic control of native enzyme expression are expected to solve the problem –77. DynaThe increasingly sophisticated synthetic biology and metabolic engineering toolbox is already having an impact on the number and frequency of reported successful whole-cell biocatalytic processes. This field will thus be highly dynamic for the foreseeable future.


The ACS group was administered 1.0 g of anserine/carnosine (3:1) for 3 months. A microarray analysis and subsequent quantitative real-time polymerase chain reaction (qRT-PCR) analysis of peripheral blood mononuclear cells (PBMCs) showed decreased expression of CCL24, an inflammatory chemokine (p < 0.05). Verbal memory, assessed using the Wechsler memory scale–logical memory, was preserved in the ACS group. An age-restricted sub-analysis showed significant verbal memory preservation by ACS in participants who were in their 60s  and 70s . The suppression of CCL24 expression was greatest in people who were in their 70s (p < 0.01). There was a significant correlation between the preservation of verbal memory and suppression of CCL24 expression in the group that was in the 70s . These results suggest that ACS may preserve verbal episodic memory, probably owing to CCL24 suppression in the blood, especially in elderly participants.Our goal was to determine whether anserine/carnosine supplementation (ACS) suppresses chemokine levels in elderly people. In a double-blind randomized controlled trial, volunteers were assigned to the ACS or placebo group (1:1). Sixty healthy elderly volunteers (active,  Owing to their identical chemical structures, with the exception of anserine methylation, anserine and carnosine have equivalent reported physiological functions [Carnosine is an endogenous dipeptide consisting of beta-alanine and histidine, which is present in the millimolar range in skeletal muscle and in the one hundred micromolar range in the vertebrate brain . Carnosiunctions . At presunctions  and our unctions ,9 have sIn this study, we elucidated the mechanism underlying the preservation of verbal episodic memory in elderly people, as evaluated by the Wechsler memory scale–logical memory (WMS-LM) . Normal Sixty-nine healthy volunteers (41–78 years of age) were enrolled in this study between June 2012 and November 2013 from the Tokyo metropolitan area, Japan . All subl-lysine) and 150 mg/day l-histidine, because the enzymatic digestion of carnosine (250 mg/day) generates l-histidine (150 mg/day) and beta-alanine. Both treatments were granular solids taken orally over a 3-month period. All sixty participants reported to take more than 80% of test formulae during the test period. Absorption of imidazoledipeptide in elderly people has been confirmed in a separate test (data not shown).The test formula was a powder containing anserine and carnosine (3:1) derived from chicken meat, provided by NH Foods Ltd., Ibaraki, Japan. Participants in the ACS group received doses of the imidazole dipeptide formula (500 mg/dose) twice-daily. The safety of this formula was previously verified by two independent studies ,14. The A dietary survey was conducted using a semi-quantitative method, as reported elsewhere . At the The following cognitive evaluation tools and self-report questionnaires were used to assess the effects of ACS on cognitive function, mental status, and general health: (1) the Japanese version of the Wechsler memory scale–revised logical memory immediate recall (WMS-LM1) and delayed recall (WMS-LM2) tests ; and (2)t-test was applied using the MeV software [p < 0.05) using a Student’s t-test between the Delta of placebo and active samples (exposed to ACS). One hundred and fifty-one significant genes were implicated in the “immune response” or “inflammatory response” in the gene ontology annotation.Peripheral blood mononuclear cells (PBMCs) from volunteers were used for total RNA extraction using a PAXgene Blood RNA Kit . The total RNA quality was assessed using the Agilent system according to the manufacturer’s protocol. We eliminated several RNA samples from the two test groups owing to poor quality. cRNA was labeled using low input quick amp labeling , and hybridized onto a human whole genome oligo DNA Microarray Ver2.0 , as described elsewhere . Relativsoftware . We obtacDNA was prepared using ReverTra Ace . qRT-PCR was performed using the KAPA SYBR Fast qPCR Kit  and Thermal Cycler Dice Real Time System TP-800 . Samples were analyzed in triplicate, and gene expression levels were normalized to the corresponding β-actin level. The PCR primers for CCL24 were as follows: Forward primer: 5′-CCAGCCTTCTGTTCCTTGGTG-3′; reverse primer: 5′-AACTGCTGGCCCTTCTTGGT-3′.We used the data from all participants who completed the study. The participants were randomly assigned to the ACS and placebo groups. Sixty participants completed both the baseline and follow-up tests. The group characteristics are summarized in The purpose of this study was to test the effect of anserine/carnosine supplementation, but these dipeptides are also obtained from the diet. To estimate the daily intake from the diet, we estimated the anserine + carnosine intake from animal and fish meats using a 7-item meat intake frequency questionnaire . There wt-test was conducted between the Deltas of the ACS and placebo groups, which showed that 2650 genes were significantly differentially expressed (p < 0.05). Of these, we focused on the genes (151) categorized as “inflammatory-related genes including soluble factors”, such as chemokines, which would be expected to affect neural tissues and/or cerebrovascular cells, and further selected six genes (Chemokine (C-C motif) ligand 24 (CCL24), platelet-derived growth factor subunit B (PDGFB), interleukin (IL)-17B, IL-28A, IL-4, and IL-6) with a relatively higher expression level . Of these, we selected the inflammatory chemokine CCL24 for further analysis because CCL24 showed the highest level of expression. p < 0.05).We performed a microarray analysis of the blood samples obtained from participants at the baseline and follow-up timepoints. After normalization of the gene expression between the samples, we calculated the log2 fold-change (Delta) between the pre-intake and post-intake blood samples as altered expression values. A student’s p = 0.078. In the WMS-LM1 test, used to assess the immediate recall of verbal memory, we did not see any difference between the two groups (p = 0.31). In the ADAScog test, the Time × Variant interaction was not significant (p = 0.47). In the PCS score from the SF-36, the Time × Variant interaction showed a trend towards significance (p = 0.08). In the MCS score from the SF-36, the Time × Variant interaction was not significant (p = 0.30). In the BDI test, the Time × Variant interaction showed a weak trend towards significance (p = 0.14).To evaluate cognitive function at baseline and follow-up, we performed two neuropsychological tests . For thep = 0.048). We also observed a trend towards significance in the suppression of CCL24 expression (p = 0.14). For participants in their 70s (p = 0.017) and CCL24 expression (p = 0.006) between the two groups. There was a significant correlation between the WMS-LM2 test score and the ratio of CCL24 expression . Using data from daily anserine/carnosine intake from participants, we also plotted total daily intake of the two dipeptides (anserine and carnosine) versus the ratio of CCL24 expression in participants who were in their 70s  participants in their 60s ; and (2)heir 70s , we founheir 70s . We founIn this study, we found that ACS significantly suppressed the expression of a blood inflammatory chemokine, CCL24, in elderly participants . In the CCL24 is a member of the eotaxin family (eotaxin-2) . AnotherGiven that the delayed-recall tests of verbal memory, such as the WMS-LM2 test  or the fIn this study, we detected significant decreases in CCL24 expression in the ACS group after supplementation with ACS, which agrees with our previous finding that ACS treatment decreases the production of IL-8 in elderly people , and carOur study has various limitations. The sample size was limited, and represented an age-based subgroup analysis from a larger original group that included data from younger participants. Age is an important risk factor in Alzheimer’s disease, in order to systematically defining age sensitivity/dependences we are in underway to plan next RCT study with a larger number of participants. In this study, we used a mixture of anserine and carnosine, and we did not establish which molecule was the most effective. While we tried to estimate the participants’ dietary intake of anserine and carnosine using a questionnaire, a better method is needed to estimate their levels more accurately.


Mothers living with small children in Fukushima prefecture may experience radiation anxiety and related symptoms after the Fukushima Dai’ich Nuclear Power Plant Accident. A behavioral activation (BA) program was developed to improve their psychosomatic symptoms. The purpose of this randomized controlled trial was to examine the effectiveness of a BA program for improving psychological distress and physical symptoms among mothers with preschool children in Fukushima-prefecture 3 years after the Fukushima Daiichi Nuclear Power Plant accident.Participants were recruited from mothers living with a preschool child(ren) in Fukushima city and surrounding areas though a newspaper advertisement, posters, and flyers. Participants allocated to the intervention group received a newly developed group-based BA program, which consisted of two 90- min lessons with a 1-week interval. Psychological distress and physical symptoms as a primary outcome, and radiation anxiety and positive well-being (liveliness and life satisfaction) as a secondary outcome, were measured at baseline, 1- and 3-month follow-ups.p = 0.051) and physical symptoms (p = 0.07) at 1-month follow-up, while the effect became smaller at 3-month follow-up. The effect sizes at 1-month were medium to large . There was a significant intervention effect on increasing liveliness at 3-month follow-up (p = 0.02); and there were marginally significant effects on life satisfaction at 1- and 3-month follow-ups (both p = 0.09).Participants were randomly allocated to either an intervention or a control group . The BA program showed a marginally significant intervention effect on psychological distress . Registered 27 May 2014.The UMIN Clinical Trials Registry (UMIN-CTR; ID =  On March 11, 2011, a huge earthquake hit northeast Japan, and the Pacific coast of this region suffered from a tsunami, with 15,882 casualties and 2,668 still missing. The tsunami also hit heavily the Tokyo Electric & Power Company Fukushima Dai-ichi nuclear plant. The plant lost control over regulating its nuclear reactors, which resulted in several explosions of the reactors and a discharge of radioactive substance, which flowed widely to a west-to-northwest direction from the plant. World Health Organization (WHO) and United Nations (UN) reports have estimated a cumulative effective dose in the first year following the accident for adults at 1–10 micro Sv in the sea-side area and other less affected areas of Fukushima prefecture  = 0.60 to 1.15), as compared with control conditions  to 4 [all of the time]) with which respondents reported the degree of psychological distress during the past 30 days. The total score ranges from 0 to 24. The internal reliability and validity found in previous studies are acceptable ), liveliness (1.47 [95 % CI 0.66 to 2.27]) and life satisfaction (1.19 [95 % CI 0.21 to 2.17]) at 3-month follow-up. The effect size was greater among housewives (n = 12 in each group) for life satisfaction (1.05 [95 % CI 0.32 to 1.77]) at 1-month follow-up. The effect sizes were greater among mothers who had university graduates or higher education (n = 10 in the intervention group and n = 5 in the control group) for liveliness (1.14 [95 % CI 0.21 to 2.08]) at 3-month follow-up and life satisfaction (1.56 [95 % CI 0.83 to 2.28]) at 1-month follow-up. The effect sizes were greater among mothers with two or more children for psychological distress (n = 10 in the intervention group and n = 12 in the control group) at 1-month (-1.24 [95 % CI -1.94 to -0.55]) and 3-month (-0.93 [95 % CI -1.73 to -0.14]), and for liveliness (1.12 [95 % CI 0.34 to 1.89]) at 3-month follow-up.There were a few subgroup differences observed in the effect of the BA program: the effect sizes were greater for employed mothers (N = 18) in the intervention group received the two lessons of the group-based BA program. A total of 17 (94.4 %) responded that the behavioral activation program was easy to moderately easy to understand; 17 (94.4 %) reported the program useful; and 18 (100 %) rated their satisfaction with the program as very much or mostly.All of the participants  at 1-month follow-up, with medium to large effect sizes. The BA program showed a significant intervention effect on improving a secondary outcome, liveliness, at 3-month follow-up with a large effect size. This BA program may be effective on improving psychological distress, physical symptoms and psychological well-being among mothers with preschool children after the nuclear power plant accident in Fukushima, even though a further large-scale study is required to confirm the intervention effects.To our knowledge, the present study is the first to examine whether a BA-based program could improve psychological distress and physical symptoms that were affected by radiation anxiety in a non-clinical community population. However, there were only marginally significant effects on psychological distress and physical symptoms at 1-month follow-up. A possible explanation for the lack of effect of the present BA-based intervention could be the low intensity and short duration of the intervention. A previous meta-analysis reported that the median number of clinical sessions with a therapist was eight and the number of sessions was not associated with effect size . HoweverThe present study failed to show significant effects of the BA program on improving radiation anxiety at 1- or 3-month follow-ups. However, radiation anxiety tended to improve at 1-month follow-up to some extent, as also did physical symptoms and psychological distress. A possible process of health anxiety causing hypochondriasis is that excessive anxiety about one’s health leads to believing oneself to be sick, because of misinterpretation of one’s physical symptoms as a signal of a serious health problem . The resIn the present study, liveliness significantly improved at 3-month follow-up; and life satisfaction marginally significantly improved at 1- and 3-month follow-ups. A previous meta-analysis reported that BA interventions significantly improved well-being . The preBased on the subgroup analyses, participating mothers who were employed, highly educated, or had two or more children seemed to enjoy benefits from the present BA program for a longer term . The BA program may remain more effective for employed mothers, since they may have more chance to exercise BA in a wider social network including their workplaces. The BA program may be more effective for mothers with higher education, because of their better understanding the program contents. The BA program may be more effective for mothers with multiple children. They may have more worry and burden in rearing their children under radiation anxiety, and thus feel a greater need to learn the BA program. Unfortunately, due to the small sample size, the present study could not confirm these subgroup differences, that should be fully investigated in future research.Possible limitations of the present study should be considered. A major limitation of this study was the small sample size. The sample size of the present study was too small. Preliminary sample size calculation showed that the necessary sample size was 30 participants per arm, while the present study had hardly two-thirds that number. The present study may have overlooked some effects of the BA program because of the limited statistical power. Second, participants were recruited from Fukushima city and neighboring municipalities, not from the entire aria of Fukushima prefecture. Most participants were housewives, and had a higher education. Participants in the present study may not be representative of mothers with radiation anxiety in the whole Fukushima prefecture. Therefore, the generalizability of the present findings may be somewhat limited. Third, all outcomes in the present study were measured by self-report, which may be affected by the perception of participants or by situational factors. A self-reported measure could be vulnerable to a cognitive bias. If participants answered the questionnaire in a socially desirable manner, the observed association may have been overestimated. Fourth, residence year of participants were not assessed. Fifth, follow-up period in this study was limited to estimate its long-term effect. Sixth, the present study did not examine the cost-effectiveness of the BA program. Seventh, there may be heterogeneity of the effect of the present BA program depending on educational attainment, employment status, and the number of children of participating mothers, which we could not investigated in detail in this study because of the small sample size. A further RCT should be conducted to examine whether the BA program is effective in a larger representative sample of mothers living in the whole Fukushima prefecture with a long-term follow-up.This BA program may be effective for improving psychological distress, physical symptoms, and well-being, at least for a short duration, among mothers with preschool children after the nuclear power plant accident in Fukushima, while a further large-scale study is needed. This program may possibly contribute to improve health and well-being of mothers living in Fukushima, provided by trained public health nurses at each local public health center in Fukushima prefecture.


Furthermore, we assessed the possible involvement of Wnt/β-catenin pathway in response to HP. Chondrocytes were exposed to HP for 3h and the evaluations were performed immediately after pressurization, and following 12, 24, and 48 h. Total RNA was extracted and used for real-time PCR. β-catenin was detected by Western blotting analysis and immunofluorescence. In OA chondrocytes, HP induced a significant increase (p < 0.01) of the expression levels of miR-27a/b, miR-140, and miR-146a, and a significant reduction (p < 0.01) of miR-365 at all analyzed time points. MMP-13, ADAMTS-5, and HDAC-4 were significantly downregulated following HP, while no significant modification was found for IGFBP-5. β-catenin levels were significantly increased (p < 0.001) in OA chondrocytes at basal conditions and significantly reduced (p < 0.01) by HP. Pressurization did not cause any significant modification in normal cells. In conclusion, in OA chondrocytes, HP restores the expression levels of some miRNAs, downregulates MMP-13, ADAMTS-5, and HDAC-4, and modulates the Wnt/β-catenin pathway activation.Mechanical loading and hydrostatic pressure (HP) regulate chondrocytes’ metabolism; however, how mechanical stimulation acts remain unclear. MicroRNAs (miRNAs) play an important role in cartilage homeostasis, mechanotransduction, and in the pathogenesis of osteoarthritis (OA). This study investigated the effects of a cyclic HP (1–5 MPa), in both normal and OA human chondrocytes, on the expression of  Osteoarthritis (OA) is a chronic degenerative joint disease characterized by degradation of articular cartilage, synovial inflammation, thickening of subchondral bone, and formation of osteophytes .The major feature of OA is the destruction of extracellular matrix components (ECM) due to the imbalance towards catabolic activities of cartilage-degrading enzymes, like matrix metalloproteinases (MMPs) and disintegrin and metalloproteinase with thrombospondin motif (ADAMTS) . The patMechanical force plays a fundamental role in regulating cartilagemorphogenesis and maintenance.It has been demonstrated that cycles of loading, at which articular cartilage is physiologically subjected, are fundamental to regulate metabolic activity of chondrocytes, while an injuring overload is responsible for their damages and death ,4.A particular mechanical stress that acts in vivo is hydrostatic pressure (HP). Several in vitro studies have demonstrated that HP works as a modulator of cartilage metabolism , influenRecent studies have underlined the key role of microRNAs (miRNAs) in regulating chondrocyte functions ,10. miRNMMP-13 and insulin-like growth factor binding protein (IGFBP)-5 expression levels [miR-27a and miR-27b seem to be among the main studied miRNAs involved in OA; recent evidence has been reported that their lower expression in OA cartilage in comparison to normal cartilage was accompanied bydysregulation of both n levels ,18.MMP-13, ADAMTS-5, and IGFBP-5 [miR-140 could be considered a regulator of chondrocyte differentiation, bone development, and cartilage homeostasis. It has been demonstrated that a reduced expression of this miRNA in OA cartilage influenced the expression levels of its target genes  IGFBP-5 ,19,20.Many studies suggest the involvement of levels of miR-146a and miR-146b in the development and progression of OA, even though their expression in OA cartilage and their specific function has not yet been fully understood ,22.(HDAC)-4. Furthermore, miR-365 is a mediator of inflammatory response [miR-365 is also correlated with cartilage catabolic processes in OA: its upregulation in OA chondrocyte cultures is induced by mechanical overloading, through the inhibition of its target gene histone deacetylase response .The biochemical and molecular mechanisms by which mechanical stress influences transcriptional activities in chondrocytes remain unclear. It has been reported that the Wnt/β-catenin pathway is associated with mechanical force application in joints, and that this signaling is activated after mechanical damage to cartilage ,25.The WmiR-27a/b, miR-140, miR-146a/b, and miR-365 expression levels, as well as on the mRNA levels of their target genes, MMP-13, ADAMTS-5, IGFBP-5, and HDAC-4, in human normal and OA chondrocyte cultures. Furthermore, we evaluated the possible activation of the Wnt/β-catenin signal pathway through Western blotting analysis and immunofluorescence in order to establish its involvement in response to HP.The purpose of this study was to investigate the possible effect of cyclic hydrostatic pressure (HP)  on MTT test was performed to evaluate the effect of HP on chondrocyte survival. The mechanical loading tested in the study did not affect cell viability, neither immediately after HP nor at the various considered time points, in OA and normal chondrocytes (data not shown).These data were confirmed by a trypan blue test (data not shown).miR-27a, miR-140, and miR-146a were significantly downregulated in OA cell cultures in comparison to normal (p < 0.01), while miR-365 was upregulated in OA chondrocytes (p < 0.01). Concerning miR-27b and miR-146b, no significant differences between OA and normal cells were detected . On the contrary, IGFBP-5 gene expression appeared to be significantly downregulated in OA in comparison to normalchondrocytes, as evaluated at basal time (p < 0.01)  B.miR-27a/b, miR-140, and miR-146a/b in OA chondrocytes (p < 0.01) immediately after pressurization (T0), compared to the unloaded controls. At different time points following HP application , high significant levels of miR-27a, miR-140, and miR-146a gene expression were maintained (p < 0.01) in comparison to the corresponding load-free conditions. The same results were obtained analyzing miR-27b levels, except for T48. The effect of loading on miR-146b increase was only restricted to the T0 time point. Concerning miR-365, our data showed its significant reduction after pressurization at the different analyzed time points in OA cells (p < 0.01). No significant modifications of all the analyzed miRNAs were observed over time in normal cells , as well as at the different analyzed time points compared to the respective unloaded controls (p < 0.01). On the contrary, IGFBP-5 expression showed no significant modifications in response to HP. No significant changes in the evaluated target genes were observed over time in normal cells.p < 0.001) β-catenin protein levels in OA samples, in comparison to normal, at basal conditions. Immediately after 3 h of HP exposure, a significant reduction of protein levels in OA chondrocytes was induced (p < 0.01), while no detectable differences were observed in normal cells following pressurization .miR-27a/b, miR-140, miR-146a/b, and miR-365 expression levels, which are responsible for the regulation of mRNA levels of their target genes, MMP-13, ADAMTS-5, IGFBP-5, and HDAC-4, in normal and OA chondrocyte cultures.The aim of this study was to evaluate if a cyclic HP could influence cell transcriptional activity, modifying One of the main factors regulating the metabolic activity of chondrocytes is the mechanical stimulus; articular cartilage is constantly subjected to loading cycles that depend on body weight, muscle tension, posture, and physical activity ,28.The typical composition and organization of cartilage extracellular matrix is maintained in a state of constant turnover by a balance of anabolic and catabolic activities of the chondrocytes, providing osmotic properties necessary for the ability of the tissue to resist compressive stresses .Pressure levels on joints subjected to load have been measured in vivo; a pressure magnitude of 5 MPa is most often encountered in the knee joint during a normal gait . In thisOur results showed that HP was able to modulate the expression levels of some miRNAs involved in the pathogenesis of the disease, in OA chondrocytes but not in normal cells. In fact the data confirmed previous evidence reporting that no changes occur when normal chondrocytes are subjected to pressure close to the physiological range of the human joint ,31.miR-27a downregulation. There is no evidence about the possible effects of mechanical loading on modification of miR-27 a/b. Our study, for the first time, showed a positive effect of HP in OA cells, restoring the miRNA levels observed in normal conditions. This positive effect could be detected not only immediately after HP exposure, but was also maintained over time until 48 h.The role of miR-27a/b in OA has been pointed out; in fact, their gene expression was reduced in OA chondrocytes in comparison to normal ,18, as cMMP-13 and IGFBP-5 were its target genes, as predicted by bioinformatic approaches [miR-27b and its target gene MMP-13 in an in vitro study of human OA chondrocytes [Transfection of OA chondrocytes with anti-miRNA specific for miR-27a confirmed that proaches . In addidrocytes . It is wdrocytes ; in partdrocytes .IGFBP-5 is a protein that constitutes a reservoir of the anabolic factor IGF-1, promoting aggrecan and collagen fiber deposition and cell proliferation. Clemmons et al. showed that a downregulation of this protein in OA cartilage, associated with a reduced availability of IGF-1, contributed to extracellular matrix degradation , even thMMP-13 in OA chondrocytes at basal conditions in comparison to normal cells; these expression levels were reduced following HP and were then maintained until 48 h after the application of mechanical loading. Interestingly, our data confirmed that IGFBP-5 mRNA levels were decreased in OA cells at baseline according to Tardif et al. [As expected, our results reported a significant production of f et al. . Howeverf et al. .miR-140 regulation remained unknown. A significant increase of this miRNA level was induced by our pressurization system at all the analyzed time points, showing that miR-140 acts as a mechanical transduction modulator. The positive effect of miR-140 upregulation is also accompanied by a significant decrease in the expression levels of MMP-13 and ADAMTS-5, well known as matrix-degrading enzymes implicated in cartilage degradation during OA damage [MMP-13, ADAMTS-5, and IGFBP-5 are miR-140 direct target genes [IGFBP-5 3’-UTR is also targeted by miR-140, which plays an important role in OA pathogenesis; in fact, as shown by several studies on chondrocyte cultures, its expression was downregulated in injured cells, especially after pro-inflammatory cytokine stimulation ,20. The A damage ,37,38. Tet genes ,39.miR-146a, but not of miR-146b, in OA cells. Current knowledge about the expression trend of these miRNAs reported contrasting data. Li and colleagues affirmed that miR-146a is overexpressed in OA [miR-146a was intensely expressed in low-grade OA cartilage in comparison to normal tissue, but resulted progressively downregulated in the late stage of the disease, suggesting a potential role of this miRNA as a promoter of OA inflammation [miR-146a and a simultaneous activation of apoptosis in human normal chondrocyte cultures. Furthermore, Jin et al. observed, for the first time, that this miRNA plays a mechano-responsive activity, in a time-dependent manner [miR-146a appeared to be upregulated following HP and this effect was maintained until 48 h. The obtained data seem to be in contrast with the current literature; however, this can be explained by the different experimental conditions applied, such as the cellular type analyzed and entity of loading, both for the intensity and for the duration. Recently, transfection experiments on human chondrocytes showed a negative regulation of miR-146a on MMP-13 and ADAMTS-5 levels, suggesting its anti-catabolic property [MMP-13 and ADAMTS-5 in OA cultures at basal conditions and a significant reduction of their expression was obtained after the exposure of HP at all the analyzed time points.In our study, the comparison between normal and OA chondrocytes at basal conditions, showed a significant downregulation of ed in OA , while Jammation ,21. It wproperty . Our datmiR-146b neither in OA human chondrocyte culture nor after the application of mechanical loading. In the current experiment HP increased the expression levels of miR-146b only at the end of pressurization (T0); however, our experiment does not allow us to explain this result.There was no evidence regarding the evaluation of the expression levels of miR-365 in OA chondrocyte cultures compared to normal cells. The upregulation of miR-365 contributed to an abnormally accelerated differentiation and proliferation of hypertrophic chondrocytes and inflammatory cytokine production, increasing catabolic effects [miR-365 was upregulated by an injuring mechanical loading, and by the stimulation with pro-inflammatory cytokines [miR-365 gene expression was significantly reduced in OA cultures.The present study confirms previous data reported by Yang et al. showing the overexpression of  effects . In addiytokines . FollowiMMP-13, in human OA cartilage explants and chondrocytes [HDAC-4 was identified as a direct target gene of miR-365 [HDAC-4 is a protein that plays a critical role in transcriptional regulation, cell cycle progression, and developmental events. It is a major regulator of cartilage development and endochondral ossification, functioning as a potent inhibitor of chondrocyte hypertrophy and differentiation . Interesdrocytes . Followi miR-365 .miR-365 at mechanical loading, the HDAC-4 expression levels modulation after pressurization was expected. In fact, our HP induced the HDAC-4 down-regulation, decreasing the catabolic activities of chondrocytes associated to OA pathogenesis [miR-365 after HP, should be associated by an increase of HDAC-4 mRNA. However, Guan and coll. reported that miR-365 regulated the protein levels of HDAC-4, but not its mRNA levels [HDAC-4.Since the responsiveness of ogenesis , even atA levels . Our eviThe canonical Wnt signaling pathway plays a fundamental role in embryonic skeletal development and articular cartilage growth and homeostasis ,47,48. TThe important role of Wnt signaling in regulating miRNAs expression has been reported ,52. In aWe analyzed β-catenin protein levels and cell localization after 3 h of HP, in agreement with a previous report suggesting that translocation of β-catenin is a rapid process ; in factNormal human articular cartilage was aseptically obtained from post-traumatic femoral heads of five subjects  with no known history of joint disease; macroscopic and microscopic analysis were performed to confirm that the tissue used was normal. OA human articular cartilage was obtained from the femoral heads of five patients  with hip OA defined by clinical and radiological American College of Rheumatology criteria , undergog. The trypan blue viability test pointed out a 90%–95% cell survival for both normal and OA chondrocytes. Cells were incubated for two weeks at 37 °C and 5% CO2 in DMEM culture medium containing 10% fetal calf serum, 200 U/mL penicillin, and 200 µg/mL streptomycin. The medium was changed three times per week. The cell morphology was examined daily with an inverted microscope  to avoid the dedifferentiation of expanded chondrocytes and preserve their phenotypic stability.Following surgery, the cartilage was aseptically dissected and minced into small pieces. The fragments were washed in DMEM with phenol red, containing 2% penicillin/streptomycin solution and 0.2% amphotericin B. The chondrocytes were isolated from the articular cartilage using sequential enzymatic digestion: 30 min with 0.1% hyaluronidase, 1 h with 0.5% pronase, and 1 h with 0.2% collagenase at 37 °C in the wash solution (DMEM + penicillin/streptomycin solution + amphotericin B). The resulting cell suspension was filtered twice using 70-μm nylon mesh, then washed and centrifuged for 5 min at 700× All experiments were performed by using three replicates of cell cultures from each single donor.5 cells re-suspended in 2 mL of medium with phenol red containing 10% fetal calf serum, 200 U/mL penicillin, 200 U/mL streptomycin, and 2 mM glutamine until they became 85%–90% confluent. The primary single layer of chondrocyte cultures was evaluated before starting the experiment  and after the cyclic HP exposure.In the first passage normal and OA human chondrocytes were seeded in Petri dishes (35 × 10 mm) at a starting density of 1 × 10The pressure used for this study was generated by our prototype of pressurization system, provided by a hermetically sealed pressure chamber . Petri dExperiments have been performed using chondrocytes and supernatants collected at basal conditions and immediately after receiving pressure (T0), as well as after 12 h (T12), 24 h (T24), and 48 h (T48) of retention in culture, following pressurization. Some dishes, maintained in the same culture conditions for the same period of time without receiving any pressurization, were used as controls.Cell viability was evaluated at various time points named above by 3--2,5-Diphenyltetrazolium Bromide MTT assay. Chondrocytes were in cubated for 3 h at 37 °C in a culture medium containing 10% of 5 mg/mL MTT  . The medium was then discarded and 0.2 mL of DMSO  was added to each well to solubilize the formazan crystals. The absorbance was measured at 570 nm using a microplate reader . Blank measurement was performed using a well without cells.The percentage of cell survival was calculated as follows:4 adherent cells.The experiments were carried out on pre-confluent cell cultures to prevent contact inhibition influencing the results. Data were expressed as OD units per 10Total RNA, including miRNA, was extracted using TriPure Isolation Reagent according to the manufacturer’s instructions and was stored at −80 °C. The concentration, purity, and integrity of RNA were evaluated by measuring the OD at 260 nm and the 260/280 and 260/230 ratios by Nanodrop-1000 . The quality of RNA was also checked through electrophoresis on agarose gel . Reverse transcription for miRNA was performed using the cDNA miScript PCR Reverse Transcription , while that for target genes was done using Quanti Tect Reverse Transcription Kit , according to the manufacturer’s instructions.MiRNAs and target genes were analyzed by real-time PCR using, respectively, miScript SYBR Green  and Quanti Fast SYBR Green PCR (Qiagen) kits and the primers listed in To further analyze the dissociation curves, we visualized the amplicons’ length in an agarose gel to confirm the correct amplification of the resulting PCR products.Ct values in each sample and the efficiencies of the primer set were calculated using LinReg Software [For data analysis, the Software  and thenSoftware .Normalization was carried out considering as housekeeping genes SNORD-25 for miRNAs, and ACTB for target genes. These genes were chosen, analyzing genes listed in 5 cells/chamber and re-suspended in 2 mL of culture medium until they became 85%–90% confluent. Some dishes were subjected to 3 h of HP, while control dishes were maintained in the same culture conditions for the same period of time without receiving any pressurization. The samples were collected immediately after 3 h of HP to evaluate the β-catenin activation, in agreement with Niu et al. [Normal and OA chondrocytes at the first passage were seeded in Petri dishes (35 × 10 mm) at a starting density of 1 × 10u et al. . Total c4 cells/chamber, to avoid possible cell overlapping, and re-suspended in 2 mL of culture medium until they became confluent. Some dishes were subjected to 3 h of HP, while others were used as controls. The cells were processed after 3 h to further confirm the pathway activation. The chondrocytes were rinsed twice in phosphate-buffered saline (PBS) and fixed in methanol for 20 min and in acetone for 5 min at −20 °C. The samples were then treated with a blocking solution , 5% normal goat serum (NGS)) for 20 min at room temperature and incubated overnight at 4 °C with mouse monoclonal anti-β-catenin (12F7) antibody (Santa Cruz Biotechnology) diluted 1:50 in PBS, 0.1% BSA, and 1% NGS. After three washes in PBS, the samples were treated with goat anti-mouse IgG-Texas Red conjugated antibody  diluted 1:100 in PBS, 0.1% BSA, and 1% NGS. Finally, the samples were washed three times in PBS and mounted with Vecta shield . Negative controls were obtained by omitting the primary antibody. Observations were carried out with a Leitz Aristo plan fluorescence microscope. We randomly selected five visual fields for each sample and calculated the percentage of the number of cells exhibiting nuclear translocation of β-catenin to the total number of cells in each field of view [Normal and OA chondrocytes at the first passage were seeded on coverslips in Petri dishes (35 × 10 mm) at a starting scarce/low density of 8 × 10 of view . A totalThe results were expressed as the mean ± standard deviation of triplicate values obtained from the experiments derived by each single donor. Data normal distribution was confirmed by Shapiro–Wilk, D’Agostino–Pearson, and Kolmogorov–Smirnov tests.Ct−ΔΔ values for each sample. Western blot data were analyzed by ANOVA with a Bonferroni posthoc test. Immunofluorescence staining was scored by the same researcher and the results were analyzed by chi-square test. All analyses were performed using the SAS System  and GraphPad Prism 6.1 .A significant effect was indicated by a p-value <0.05.Real-time PCR were evaluated by one-way (ANOVA) with a Tukey’s post hoc test using 2Our results demonstrated for the first time the effect of a cyclic HP that approximates the physiological condition of human joints in restoring the expression levels of some miRNAs normally dysregulated in OA. Moreover, a significant reduction of transcriptional levels of the principal proteases responsible for OA cartilage matrix degradation was also detected. The positive influence of pressurization was observed immediately after loading and maintained over time, until 48 h. The observed transcriptional modifications of miRNAs and target genes in OA chondrocytes could be caused by HP-induced Wnt/β-catenin signaling pathway modulation.These data suggest that physiological loading is able to influence the epigenetic regulation of chondrocyte homeostasis; this leads us to consider moderate physical exercise, in clinical practice, as a useful approach to limit articular cartilage damage in OA. Nevertheless, some study limitations need to be mentioned. Firstly, transfection experiments with specific anti-miRNA could be useful to confirm the regulation of target genes expression by the analyzed miRNAs. In addition, target genes’ protein levels should be detected to elucidate if transcriptional changes also reflect a translational regulation. Finally, experiments with specific Wnt/β-catenin inhibitors are recommended to confirm the role of this pathway in mediating the response to HP.